Figure 1. Involvement of GRK6 in the engulfment of apoptotic cells.

(a) NIH3T3 cells were infected with the empty retrovirus vector or the virus carrying the cDNA for kinase-inactive form of GRK2, GRK3, GRK5 or GRK6 (GRK2 (K220R), GRK3 (K220R), GRK5 (K215R) or GRK6 (K215R)) with N-terminal Flag tag. The infected cells (4 × 10⁴ cells) were co-cultured with CMFDA-labelled WT thymocytes (1 × 10⁶ cells) at 37 °C for 90 min. Percentages of the phagocytes carrying the engulfed thymocytes were determined by FACS as detailed in Methods. The representative FACS profiles with the NIH3T3 cells expressing GRK2 (K220R), GRK3 (K220R), GRK5 (K215R) or GRK6 (K215R) are shown from four independent experiments performed in triplicate. The expression level of each GRK mutant in NIH3T3 cells was determined by immunoblot analysis using anti-Flag and anti-GAPDH antibodies. (b) The engulfment ability of NIH3T3 cells transfected with control or GRK6 siRNA was estimated by FACS analysis as described in a. The cell lysates of the NIH3T3 cells were analysed by western blot with antibody against GRK6 or GAPDH. Expression level of GRK6 was decreased to about 30% of the normal level following treatment with GRK6 siRNA. (c) The engulfment ability of NIH3T3 cells expressing GRK6 (WT) or GRK6 (K215R) was evaluated by FACS as described in a. The level of GRK6 (WT) or GRK6 (K215R) was more than 10-fold greater than endogenous levels, determined by immunoblot using anti-GRK6 antibody. (d) BMDMs (1 × 10⁵ cells) from WT and GRK6-deficient mice were co-cultured with CMFDA-labelled apoptotic thymocytes (1 × 10⁶ cells) at 37 °C for 60 min. The percentage of the phagocytes carrying the engulfed thymocytes is presented. BMDM lysates from WT and GRK6-deficient mice were analysed by western blot with antibody against GRK6 or GAPDH. (e) CMFDA-labelled apoptotic thymocytes (8 × 10⁷ cells) were injected to WT and GRK6-deficient mice from tail vein. Two hours after the injection, their spleens were isolated and stained with anti-CD11b antibody. Percentage of CD11b⁺ splenic macrophage carrying the labelled thymocytes was evaluated by flow cytometer. All the experiments were done at least three times; all graphs show average with s.e.m. ***P<0.001. KO, knockout.